Environ. Sci. Technol. 2010, 44, 3533–3538
Polychlorinated Naphthalenes (PCNs): Congener Specific Analysis, Occurrence in Food, and Dietary Exposure in the UK A L W Y N F E R N A N D E S , * ,† DAVID MORTIMER,‡ MARTIN GEM,‡ FRANKIE SMITH,† MARTIN ROSE,† SEAN PANTON,† AND MELANIE CARR† Food and Environment Research Agency, Sand Hutton, York YO41 1LZ U.K., and Food Standards Agency, Aviation House, 125 Kingsway, London, WC2B 6NH U.K.
Received November 30, 2009. Revised manuscript received March 3, 2010. Accepted March 4, 2010.
Information on the occurrence of toxicologically significant polychlorinated naphthalenes (PCNs) in food, or on human exposure, is sparse. In this work, PCN congeners (PCNs 52, 53, 66/67, 68, 69, 71/72, 73, 74, and 75) were selected for analysis, based on the available literature on current occurrence and toxicology, and limited by the commercial availability of reference standards. The analytical methodology used cold solvent extraction of prehydrolyzed samples fortified with internal standards (13C10 labeled PCNs), activated carbon and basic alumina purification, and measurement by HRGC-HRMS. The investigation showed PCN occurrence in all studied foods: meat, milk, fish, dairy and meat products, eggs, poultry, vegetables, fruits, etc. The most frequently detected congeners were PCN 52, PCNs 66/67, and PCN 73. The highest concentrations were observed in fish (maximum value of 37 ng/kg w.w. for the sum of the measured congeners). The dioxin-like toxicity (PCN TEQ) associated with these concentrations is 1-2 orders of magnitude lower than those reported for chlorinated dioxins or PCBs in food and, on the basis of dietary intakes estimated using very conservative assumptions regarding concentrations of these contaminants in the UK, the levels of PCNs alone in food do not suggest any toxicological concerns.
Introduction Polychlorinated naphthalenes (PCNs) are chlorinated polycyclic aromatic hydrocarbonssa potentially vast group of little-known environmental contaminants of anthropogenic origin. PCNs are the most studied subgroup of these compounds and some of the 75 congeners have recognized toxic, bioaccumulative, and persistence properties which, coupled with the similarity in structural planar configuration to some PCBs and dioxins, can bestow a dioxin-like mode of toxic action. PCNs are an industrial chemical, produced over most of the last century, although manufacture is currently banned in some countries. They were sold as technical mixtures (e.g., Halowax in the U.S., Nibren in Germany, Seekay in the UK, * Corresponding author e-mail: fera.gsi.gov.uk. † Food and Environment Research Agency. ‡ Food Standards Agency. 10.1021/es903502g
alwyn.fernandes@
Published 2010 by the American Chemical Society
Published on Web 03/26/2010
etc.) of the commercial PCN product in mineral oil. The mixtures differed in the percentage of chlorine present on the naphthalene molecule, ranging from monochloro- to octachloro-substituted naphthalene. Apart from widespread commercial use as dielectrics, PCNs were also used as lubricants, electric cable insulation, paper and fabric preservatives, and plastizers. However, PCNs can also be formed through industrial thermodynamic processes such as incineration, and formation pathways resulting from de novo synthesis during combustion have been documented (1, 2). The halogenated fused diaromatic structure provides strong chemical stability and the molecule is resistant to attack by strong acids. PCN mixtures show high thermal stability, good weather resistance, good electrical insulating properties, and low flammability. However, this physical and chemical stability is responsible for the environmental persistence of the compounds. All chloronaphthalene congeners are planar and lipophilic compounds, structurally similar to the highly toxic 2,3,7,8tetrachlorodibenzo-p-dioxin molecule, and can potentially contribute to an aryl hydrocarbon (Ah) receptor-mediated mechanism of toxicity, including a combination of toxic responses such as mortality, embryotoxicity, hepatotoxicity, immunotoxicity, dermal lesions, teratogenicity, and carcinogenicity (3–7). The induction of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-de-ethylase (EROD) enzymes is a short-term biological response that is specifically indicative of planar diaromatic halogenated hydrocarbons such as dioxin and dioxin-like compounds. The few available data indicate that several PCNs are potent inducers of H4IIEEROD,AHH,andH4IIE-luc(luciferase)(4–6).Structure-activity relationships were observed in terms of the degree of chlorination and the positions of chlorine substitution. Among the PCN congeners tested, the most potent EROD, AHH, and luciferase inducers were hexa- and heptachloronaphthalenes and to a lesser extent, pentachloronaphthalenes. In humans, severe skin reactions (chloracne) and liver disease have both been reported after occupational exposure to PCNs. Other symptoms found in workers include cirrhosis of the liver, irritation of the eyes, fatigue, headache, anemia, hemeaturia, and nausea. Workers exposed to PCNs also had a slightly higher risk of all cancers combined (8). Despite the recognition of their toxic potential, very little is know about PCN occurrence and exposure, particularly of individual compounds. This is mainly due to analytical inaccessibility, caused by the lack of selective and sensitive analytical methodology, and a lack of reliable individual congener standards. Recently, some have become commercially available, and given the rising interest it is expected that more will follow. PCNs have been detected in several environmental compartments including biota. They have been measured in fish from the Great Lakes, in species such as trout, carp, bass, and pike, from low to sub-ppb levels of total PCN (9). Fish from the Detroit river showed concentrations of up to 31.4 µg/kg (9, 10) while harbor porpoises from the west coast of Sweden showed concentrations of up to 730 ng/kg ww in blubber, nuchal fat, and liver (11). A range of fish species from the Baltic sea and three Finnish lakes were measured with levels ranging from 1 to 170 ng/kg ww for Baltic sea samples and 2 to 66 ng/kg ww for the lakes (12). A study of seafood from China (13) reported levels of 0.3 ng/kg in crabs to 15.3 ng/kg whole weight in fish, for the sum of PCN homologue groups. Another recent study reports the measurement of PCNs in a range of foods, randomly acquired in seven cities of Catalonia, Spain (14). The highest concentration of total PCNs was found in oils and fats (447 ng/ VOL. 44, NO. 9, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 3533
kg), followed by cereals (71 ng/kg), fish and shellfish (39 ng/ kg), and dairy products (36 ng/kg). In general, tetra-CN was the predominant homologue group in all foods except for fruits and pulses, which had greater proportions of hexaCNs. The high levels observed in oils and fats were not reflected in vegetable or meat based products. Foods that were subject to some measure of processing (oils and fats, cereals, dairy products, meat and meat products), and fish showed the highest levels. An exposure assessment based on the data indicated that children showed higher exposure (1.65 ng/kg bw/day) than adults (0.54 ng/kg bw/day). This study developed and validated analytical methodologies for the determination of selected individual PCN congeners to investigate the occurrence of these contaminants in a range of foods, in order to establish current background concentrations and to estimate the extent to which populations are exposed through the diet.
Experimental Section Sampling and Sample Preparation. For this preliminary investigation of the occurrence of PCNs, a small number of samples of various commonly consumed individual foods, weighted toward fish and animal products, were obtained. Most samples were analyzed individually, but a fewsvegetables, fruit, and breadswere analyzed as composites of subsamples. The samples included different types of fish, meat, milk and dairy products, eggs, vegetables, etc. Samples were collected during 2007 and stored at -20 °C until analyzed. Dry solids and oils were homogenized prior to extraction by roller-mixing, milling, or blending. “Wet” samples and liquids were homogenized by blending and freeze-dried prior to extraction. Large volumes of homogenized liquids such as milk were divided into smaller (250 mL) volumes in crystallization dishes covered with ventilated aluminum foil to expedite the freeze-drying process. After freeze-drying the sub samples were rehomogenized by blending or rollermixing. Fat determinations were performed by a UKAS (ISO 17025) accredited laboratory on subsamples of the freezedried and homogenized samples, using a standard method (15). Analytes, Reagents, and Standards. The following PCN analytes were determined: PCN 52 (1,2,3,5,7-pentaCN); PCN 53 (1,2,3,5,8-pentaCN); PCN 66/67 (1,2,3,4,6,7-hexaCN/ 1,2,3,5,6,7-hexaCN); PCN 68 (1,2,3,5,6,8-hexaCN); PCN 69 (1,2,3,5,7,8-hexaCN); PCN71/72 (1,2,4,5,6,8-hexaCN/1,2,4,5,7,8hexaCN); PCN 73 (1,2,3,4,5,6,7-heptaCN); PCN 74 (1,2,3,4,5,6,8heptaCN); and PCN 75 (octachloro-CN). 13 C10 labeled standards (PCNs 42, 52, 64, and 75, and 13C12 labeled PCBs 77 and 202) were used for internal and sensitivity standardization. Reference standards for the analytes as well as the 13C10 labeled compounds were obtained from Cambridge Isotope Laboratories (Andover, MA) or from Wellington Laboratories (Guelph, Ontario, Canada) and were used after serial dilution in n-nonane. Solvents, including n-hexane, dichloromethane, nnonane, and toluene were obtained as doubly distilled grade from Rathburns Chemicals Ltd., Walkerburn, Scotland, UK. Other reagents were obtained as follows: Basic alumina was from Sigma-Aldrich, grade Super1, Type WB-5 (activated at 400-450 °C for minimum 16 h). Reagent-grade sulphuric acid and anhydrous sodium sulfate were obtained from Fisher Scientific, Loughborough, UK. Silica was obtained as 63-210 µm particle size grade from YMC Gel, Kyoto, Japan, and deionized water was generated within the laboratory. Extraction and Purification. An aliquot of the prepared, homogenized sample (equivalent to 2-5 g of lipid weight) was fortified with a known amount (200 pg of each compound) of 13C10 labeled PCN internal standard mix. The fortified sample was left to equilibrate for an hour and then blended with 200 mL of n-hexane and 75 g of acid modified 3534
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silica gel (prepared by roller mixing 1:1, H2SO4/silica, for minimum 6 h). The mixture was quantitatively transferred to a multilayer column (70 × 600 mm) packed from top to bottom with 30 g of sodium sulfate, 50 g of acid modified silica gel, 10 g of sodium sulfate, and silanized glass wool. The column was connected in series to a carbon column (20 × 95 mm containing 0.1 g of activated carbon dispersed on 1 g glass fiber) and an outflow reservoir. The columns were eluted with dichloromethane/n-hexane (40:60 v/v, 400 mL) and n-hexane (100 mL) to waste. The carbon column was disconnected and reverse eluted with 100 mL of toluene to yield a fraction containing the PCNs. The toluene extract was concentrated at an evaporation temperature of
